(1) Role of Energy-dependent Proteases in Protein Quality Control and Cell Regulation. All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. Programmed degradation of regulatory factors also contributes to controlling the cell cycle and to generating peptides for immune presentation. These activities are all carried out by energy-dependent proteases, which generically consist of two parts - a peptidase and a chaperone-like ATPase. (i) For several years, we have studied the Clp proteases of E. coli, considered as a model system. We showed that the peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpP in active complexes. We went on to show that substrate proteins bind to distal sites on the ATPase and are then unfolded and translocated axially into the digestion chamber of ClpP. In FY09, we completed a cryo-EM study of the ClpAP protease. Specifically, we reconstructed the ClpA hexamer as an integral part of the ClpAP complex. Two segments lining the axial channel were found to show anomalously low density, indicative of local mobility. By comparing a model for ClpA-ATPgS derived from cryo-EM with one built for ClpA_ADP, we infer that ATP hydrolysis is accompanied by substantial structural changes in the D2 but not the D1 tier of AAA. The entire N-domain is rendered invisible by large-scale fluctuations. When deletions of 10 and 15-residues were introduced into the linker, N-domain mobility was reduced but not eliminated and changes were observed in enzymatic activities. We also performed cryo-EM reconstructions to investigate the structure of ClpP with and without end-mounted ClpA hexamers. In the absence of ClpA, the apical region of ClpP is sealed;however, it opens up on ClpA binding, creating an access channel. This region is occupied by the N-terminal loops (residues 1 17) of ClpP. We were able to model the closed-to-open transition - which facilitates the translocation of substrates into the interior of ClpP - in terms of movements of these loops. These observations indicate that access to the ClpP degradation chamber is controlled by hinged movements of its N-terminal loops, which the binding of ClpA suffices to induce. (ii) The proteosome is responsible for ubiquitin-tagged protein degradation in eukaryotes. The 26S proteasome is composed by the 20s proteolytic chamber and the 19S regulatory complex. The 19S complex contains a hexameric ATPase ring (like ClpA - see above) and numerous regulatory proteins, of which the largest are Rpn1 and Rpn2 (both >100 kDa). It has been predicted that each has a large domain of PC repeats with alpha-solenoid folds. We confirmed this prediction in an EM analysis of both proteins. The results support the alpha-toroid model in terms of molecular dimensions and shape, and indicate that the repeats are organized not as symmetrical circular toroids, but in less regular structures. This project, first reported in FY08, was completed and published in FY09. (2) Intracellular Trafficking: Interaction of Clathrin with Proteins that Regulate its Assembly. Clathrin plays a key role in intracellular trafficking, via its coating of membranous pits and vesicles (CCVs). Assembly of clathrin is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. In the 1980s, we studied the molecular composition of coated vesicles and the plasticity of the assembly unit, the clathrin triskelion. We returned to this system in FY05, equipped with cryo-EM technology, and compared the structures of coated vesicles with and without binding of the uncoating ATPase, Hsc70. From these observations, we developed a model for uncoating. In FY07, we extended studies initiated during the previous year in which cryo-electron tomography is used to study the structures of individual CCVs isolated from bovine brain. Their polyhedral coats surround cargoes of various shapes and sizes, including vesicles containing neurotransmitters or receptors and viruses. The coated particles reconstructed in the tomograms fall into two sub-populations: 20% contain vesicles and are true CCVs;the remainder lack internal membranes and are termed "clathrin baskets" (CBs). CCVs range from 80 to 134 nm in diameter, with vesicles of 30 to 68 nm. CBs range from 66 to 120 nm. While many small polyhedral forms of coat are possible in theory, many are not observed, suggesting that they are energetically disfavored. The common feature of "forbidden" polyhedra is that they have vertices with high curvature. In tomograms we see density between the clathrin N-termini and the membrane. The vesicle is always off-center relative to the coat. To further investigate the polyhedral coats, we generated a comprehensive set of fullerene polyhedra composed of 12 pentagons and 20 -60 hexagons. These were regularized and used to calculate a variety of energetic measures, such as inter-spar angles, polygon regularity and planarity, curvature and sphericity. These results are being prepared for publication. 3) The large repertoire of immunoglobulins and T cell receptors is generated by combinatorial rearrangement of an extensive array of variable (V), diversity (D), and joining (J) gene segments that are combined to encode the variable domains of the protein chains. The recombination signal sequences (RSS) that flank these gene segments are recognized, paired in a synaptic complex, and cleaved by the proteins RAG1 (110 kDa) and RAG2 (58 kDa). Most biochemical studies on the RAG proteins have been done with the core fragments, which suffice for the basic recombination reaction, because the full-length proteins are difficult to express and purify. The RAG1/2 complex catalyzes a double-strand break at the coding/signal junctions, leaving blunt cut signal ends and DNA hairpins on the ends of the flanking coding sequences. DNA cleavage is a two-step reaction.There are two types of RSS, differing in the length of the spacer (12 or 23bp) between a conserved DNA heptamer and a nonamer). The heptamer sequence borders the coding V, D, or J segment. Efficient V(D)J recombination requires one RSS of each type. To obtain structural information on the early stages of V(D)J recombination, we used negative staining electron microscopy with image classification and averaging to visualize a RAG1/2 complex with DNA containing a pair of cleaved RSS sequences. This analysis revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N-termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near to RAG1 N-termini. These images of SEC, the post-cleavage V(D)J recombinase, provide a framework for modeling RAG domains and their interactions with DNA.